Estrogenicity of agricultural runoff: A rainfall simulation study of worst-case scenarios using fresh layer and roaster litter, and farrowing swine manure.

Scientists have correlated land application of animal wastes as fertilizer with the feminization of fish. Two questions were asked. 1) Under a worst case scenario when animal waste (layer and roaster litter, or farrowing swine slurry) is applied and tilled in 24 h prior to a surface-runoff producing rainfall, will estrogenic equivalents exceed the Lowest Observable Effect Concentration (LOEC) for fish (10 ng/L)? 2) Can calcium concentrations in runoff, measured using a rapid meter-based method, be used as a sentinel of elevated estrogenic activity? In a 3-yr study wastes were surface-applied and incorporated and 24 h later, 1.5 by 3 m plots were subjected to simulated rainfall and again 1 wk. and 3 wk. later. Nutrients in runoff were also measured, and in year 1 total coliforms and E. coli. were assessed. Except for an initial preliminary test run, runoff from all plots and years never exceeded 10 ng/L E2Eq equivalent. Calcium concentrations in runoff were not related to estrogenicity, negating its use as a sentinel marker. Specific estrogens in animal waste and runoff were identified by mass spectrometry with concentrations in runoff dependant on manure source and timing of rainfall. As expected, total coliform and E. coli concentrations in runoff were increased by the application of layer litter. Concentrations of nutrients in runoff would not be expected to result in surface water concentrations higher than guidelines for protection of aquatic species. Animal wastes applied in quantities appropriate for crop nutrient requirements, tilled into the soil surface, in observance of regulations avoiding application within 24 h of a predicted rain event, should not result in estrogen levels of environmental concern.

Do environmental factors affect male fathead minnow (Pimephales promelas) response to estrone? Part 1. Dissolved oxygen and sodium chloride.

Laboratory exposures indicate that estrogens and their mimics can cause endocrine disruption in male fishes, yet while studies of resident fish populations in estrogen-polluted waters support these findings, biomarker expression associated with field versus laboratory exposure to estrogenic endocrine disruptors (EDs) often differ dramatically. Two of the environmental parameters often found to vary in dynamic aquatic ecosystems were chosen (dissolved oxygen [DO] and sodium chloride concentrations) to assess their potential impact on ED exposure. In separate experiments, male fathead minnows (Pimephales promelas) were exposed to estrone (E1) a natural ED, under either two concentrations of DO, or two concentrations of sodium chloride, in a laboratory flow-through system. Morphological and hematological parameters were assessed. While vitellogenin concentrations were elevated with exposure to estrone (29 to 390ng/L), the effect on other indices were variable. Estrone exposure altered SSC, blood glucose, hematocrit, and hepatic and gonado-somatic index in 1 of 4 experiments, while it decreased body condition factor in 3 of 4 experiments. At the concentrations tested, no main effect differences (P<0.05) were found associated with DO or sodium chloride treatments, except in one experiment low DO resulted in a decrease in secondary sex characteristic score (SSC). The combination of DO or sodium chloride and E1 altered blood glucose in one experiment each. These results indicate the variability of fathead minnow response to estrone, even within the confines of controlled laboratory conditions.

Do environmental factors affect male fathead minnow (Pimephales promelas) response to estrone? Part 2. Temperature and food availability.

Fish are subject to constantly changing environmental conditions and food availability, factors that may impact their response to endocrine disruptors (EDs). This may, in part, explain outcome discrepancies between field studies and laboratory exposures to EDs. This study assessed whether standard laboratory conditions for fish exposures adequately represent effects of ED exposure at two environmentally realistic temperatures. The impact of temperature and food availability on male fathead minnow response to estrone (E1) exposure was studied in two experiments (3×2×2 factorial design) with three E1 concentrations (range 0-135ng/L); two temperatures (18°C and 26°C, the latter the prescribed laboratory temperature), and two feeding treatments (full fed vs. 25% of full fed) in a 21-day flow-through system. Morphometric endpoints [including body condition factor, somatic index of gonad (GSI) and liver (HSI), and secondary sex characteristics (SSC)], blood parameters [hematocrit (HCT), blood glucose, cortisol, and vitellogenin (VTG) concentrations], and histology of liver and testis were determined on day 22. High E1 consistently increased VTG, though interactions among E1, temperature and/or food on liver weight, HSI, and HCT were inconsistent between experiments. High temperature impacted the greatest number of parameters, independent of E1 treatment. Three sex-linked parameters were lower at high temperature (testis weight, GSI and VTG), and in Exp. 2SSC and gonad maturity rating were lower. At 26°C, in Exp. 1 HSI and HCT decreased, and in Exp. 2 length, body and liver weight, and body condition factor were lower. Food restriction decreased GSI in Exp. 1, and blood glucose and liver weight in Exp. 2. At 26°C several parameters were altered independent of E1 exposure, including three out of four measurements of sperm differentiation. Concordance between laboratory and field investigations of the biological effects of EDs may improve if environmentally-relevant exposure conditions, especially temperature, are employed.

Estrogenic activity and nutrient losses in surface runoff after winter manure application to small watersheds.

Confined Animal Feeding Operations generate large amounts of wastes that are land-applied to provide nutrients for crop production and return organic matter to the soil. Production practices and storage limitations often necessitate that wastes be applied to frozen and snow-covered soil. Use of application setbacks have reduced concerns related to nutrient losses in surface runoff from manure, but the estrogenic activity of runoff under these conditions has not been evaluated. Therefore, we measured and sampled surface runoff when manure was applied in the winter at a rate to meet crop N needs and measured estradiol equivalents (E2Eqs) using E-Screen. In year one, six small watersheds used to produce corn were evaluated, treatments: 2 no-manure controls, 2 liquid swine manure with 30-m setbacks, and 2 turkey litter with 30-m setbacks. In addition, beef manure was applied to six frozen plots of forage. For years 2 and 3, applications were repeated on the swine manure watersheds and one control watershed. E2Eqs and nutrient concentrations generally peaked in the first runoff event after application. The highest measured E2Eq (5.6 ng L(-1)) was in the first event after swine manure application and was less than the 8.9 ng L(-1) Lowest Observable Effect Concentration (LOEC) for aquatic species and well below the concentrations measured in other studies using ELISAs to measure hormone concentrations. No runoff occurred from plots planted with forage, indicating low risk for environmental impact, and therefore plots were discontinued from study. In years 2 and 3, estrogenic activity never exceeded the Predicted No Effect Concentrations for E2 of 2 ng L(-1). When post-application runoff contained high estrogenic activity, strong correlations (R(2) 0.86 to 0.96) of E2Eq to Ca(2+), Mg(2+), and K(+) concentrations were observed, indicating under some condition these cations might be useful surrogates for E2Eq measurements.

Serum estrogenicity and biological responses in African catfish raised in wastewater ponds in Ghana.

Reuse of wastewater for aquaculture improves the efficient use of water and promotes sustainability but the potential effects of endocrine disrupting compounds including estrogens in wastewater are an emerging challenge that needs to be addressed. We examined the biological effects of wastewater-borne estrogens on African catfish (Clarias gariepinus) raised in a wastewater stabilization pond (WSP) of a functioning municipal wastewater treatment plant, a wastewater polishing pond (WWP) of a dysfunctional treatment plant, and a reference pond (RP) unimpacted by wastewater, located in Ghana. Measurements of estrogen concentrations in pond water by liquid chromatography/tandem mass spectrometry showed that mean 17 ß-estradiol concentrations were higher in the wastewater ponds (WWP, 6.6 ng/L±2.7 ng/L; WSP, 4.9 ng/L±1.0) than the reference (RP, 3.4±1.1 ng/L). Estrone concentrations were found to be highest in the WSP (7.8 ng/L±1.7) and lowest in the WWP (2.2 ng/L±2.4) with the RP intermediate (4.7±5.0). Fish serum estrogenicity assayed by E-SCREEN was significantly higher in female vs. male catfish in the RP and WSP but not in the WWP (p≤0.05). Histological examination of liver and gonad tissue showed no apparent signs of intersex or pathology in any ponds. The similarities in various measures of body indices between fish of this study and African catfish from freshwater systems suggest that aquaculture may be a suitable reuse option for treated municipal wastewater.

Bioassay of estrogenicity and chemical analyses of estrogens in streams across the United States associated with livestock operations.

Animal manures, used as a nitrogen source for crop production, are often associated with negative impacts on nutrient levels in surface water. The concentrations of estrogens in streams from these manures also are of concern due to potential endocrine disruption in aquatic species. Streams associated with livestock operations were sampled by discrete samples (n = 38) or by time-integrated polar organic chemical integrative samplers (POCIS, n = 19). Samples were analyzed for estrogens by gas chromatography-tandem mass spectrometry (GC-MS(2)) and estrogenic activity was assessed by three bioassays: Yeast Estrogen Screen (YES), T47D-KBluc Assay, MCF-7 Estrogenicity Screen (E-Screen). Samples were collected from 19 streams within small (≈ 1-30 km(2)) watersheds in 12 U.S. states representing a range of hydrogeologic conditions, dominated by: dairy (3), grazing beef (3), feedlot cattle (1); swine (5); poultry (3); and 4 areas where no livestock were raised or manure was applied. Water samples were consistently below the United Kingdom proposed Lowest Observable Effect Concentration for 17ß-estradiol in fish (10 ng/L) in all watersheds, regardless of land use. Estrogenic activity was often higher in samples during runoff conditions following a period of manure application. Estrone was the most commonly detected estrogen (13 of 38 water samples, mean 1.9, maximum 8.3 ng/L). Because of the T47D-KBluc assay's sensitivity towards estrone (1.4 times 17ß-estradiol) it was the most sensitive method for detecting estrogens, followed by the E-Screen, GC-MS(2), and YES. POCIS resulted in more frequent detections of estrogens than discrete water samples across all sites, even when applying the less-sensitive YES bioassay to the POCIS extracts.

E-Screen evaluation of sugar beet feedstuffs in a case of reduced embryo transfer efficiencies in cattle: the role of phytoestrogens and zearalenone.

The E-Screen assay was used to evaluate the estrogenicity of sugar beet by-products obtained from a dairy farm experiencing low success rates of embryo transfer. The beet tailings had ~3-fold the estradiol equivalents of the pelleted beet pulp (3.9 and 1.2 µg estradiol equivalents or E(2)Eq/kg dry matter, respectively). Whole sugar beets, sugar beet pellets, and shreds from several Midwest US locations were also evaluated by E-Screen. All pellets examined were found to have some estrogenic activity (range ~0.1-2.0 µg E(2)Eq/kg DM) with a mean of 0.46 µg/kg dry matter and median of 0.28 µg/kg dry matter. Relative E(2)Eq ranked as follows: pellets > shreds > most unprocessed roots. Using recommended feeding levels and conservative absorption estimates (10%), the estrogenic activity in the original samples could result in blood estradiol equivalents ≥ those found at estrus (10 pg/mL, cows). Chemical analyses revealed no known phytoestrogens, but the estrogenic mycotoxin, zearalenone, was found in 15 of 21 samples. Of significance to those using the E-Screen are our findings that contradict previous reports: ß-sitosterol has no proliferative effect and genistein's glucuronidated form-genistin-is equal to genistein in proliferative effect. The latter is the result of deconjugation of genistin to genistein in the presence of fetal bovine serum (determined by LC MSMS). These data show the usefulness and caveats of the E-Screen in evaluation of feedstuffs, and indicate a potential for sugar beet by-products to contain zearalenone at concentrations that may impact reproduction.

Comparing biological effects and potencies of estrone and 17ß-estradiol in mature fathead minnows, Pimephales promelas.

The presence of endocrine active compounds such as estrogens in treated wastewater effluent and their effects on aquatic life are causing concern among aquatic resource managers. In contrast to 17ß-estradiol (E2), the steroid hormone produced by all vertebrates, the biological effects of estrone (E1), one of its breakdown products are less understood, even though the aquatic concentrations of E1 are often higher than those of E2. The central hypothesis of this study was that at environmental concentrations, E1 has estrogenic effects in fish, with increased vitellogenin concentrations and decreased reproductive success in both male and female fathead minnows, as found with E2. In two replicate experiments, we exposed mature fathead minnows to three concentrations of each estrogen for 21 days in a flow-through exposure system and measured a broad suite of anatomical (body indices, histopathology), physiological (plasma vitellogenin), behavioral (nest defense), and reproductive (fecundity, fertility, hatching) endpoints. These endpoints have previously been associated with adverse effects of estrogenic exposures. While body length and weight parameters were unaltered by exposure, secondary sex characteristics exhibited an exposure concentrated-related decline in male fathead minnows. Interestingly, low concentrations of estrone (≈ 15 ng/L) enhanced the aggressiveness of male fathead minnows in a behavioral assay. Vitellogenin concentrations in male fish increased with higher concentrations of both estrogens, but remained unchanged in all female treatments. A decrease in fecundity was observed at high concentrations of E2 as compared with control minnows. These results suggest that E1, at concentrations previously found in waters receiving wastewater effluent, can have reproductive effects on fish.

Effects of liquid swine manure on dissipation of 17ß-estradiol in soil.

17ß-estradiol (E2), a natural estrogenic hormone, degrades within hours and bind strongly to soils and sediments; however, estrogens are frequently detected in the environment at concentrations that impact water quality. Colloidal (COC) and dissolved (DOC) organic carbon may enhance the persistence and mobility of E2. Soil batch experiments were used to identify the persistence and sorption of radiolabeled E2 dissolved in solutions of (i) COC/DOC derived from liquid swine manure and (ii) CaCl(2). Estradiol disappeared from the aqueous phase before 7 d in the CaCl(2) solution, yet persisted throughout the duration of the 14 d experiment in the liquid manure solution. There was also concomitant formation of estrone (E1; a metabolite of E2) as E2 dissipated in sterile batch experiments, which was attributed to abiotic oxidation. The liquid manure solution appeared to interact with the estrogen and/or oxidation reaction sites, reducing E2 degradation. Furthermore, the liquid manure solution reduced E2/E1 binding to the soil surface resulting in more E2/E1 in the aqueous layer compared to the CaCl(2) solution. Ultrafiltration results of liquid manure indicated that ∼1/3 of E2 was associated with COC, which may be responsible for the reduced degradation and sorption of E2 in the liquid manure solution.

Comparative biological effects and potency of 17α- and 17ß-estradiol in fathead minnows.

17ß-Estradiol is the most potent natural estrogen commonly found in anthropogenically altered environments and has been the focus of many toxicological laboratory studies. However, fewer aquatic toxicological data on the effects of 17α-estradiol, a diastereoisomer of 17ß-estradiol, exists in the literature even though it has been found in the aquatic environment, sometimes at higher concentrations than 17ß-estradiol. The central objective of this study was to determine how the anatomical, physiological, and behavioral effects of exposure to 17α-estradiol compare to the well-documented effects of 17ß-estradiol exposures in aquatic vertebrates. A 21-day flow-through exposure of mature male and female fathead minnows to three concentrations each of 17α- and 17ß-estradiol (averaged measured concentrations 27, 72, and 150 ng/L for 17α-estradiol, and 9, 20, and 44 ng/L for ß-estradiol, respectively) yielded significant, concentration-dependent differences in plasma vitellogenin concentrations among estradiol-exposed males when compared to fish from an ethanol carrier control. Interstitial cell prominence in the testis of fish was elevated in all estradiol treatments. Aggressiveness of male fish to defend nest sites appeared depressed in many of the higher concentration estradiol treatments (albeit not significantly). No clear effects were observed in female fish. Based on plasma vitellogenin data, it appears that 17ß-estradiol is 8-9 times more potent than 17α-estradiol and that the lowest observable effect concentration (LOEC) for 17α-estradiol in fathead minnows is greater than 25 ng/L and may be less than 75 ng/L.

Effects of clenbuterol on body stores of polychlorinated dibenzofurans (PCDF) and dibenzo-p-dioxins (PCDD) in rats.

Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF), persistent pollutants that accumulate in the food chain, pose a risk to humans through consumption of tainted livestock. Clenbuterol, a leanness-enhancing agent, was tested for usefulness in PCDD/F body store reduction through body fat reduction (the predominant site of accumulation). To mimic the situation of contaminated animals, rats were given feed with or without a mixture of PCDD/F (0.6 to 2.7 ng/congener per day) for 10 d, followed by 16 d of feed with or without dietary clenbuterol (2 mg/kg feed). Clenbuterol reduced body fat by 28% (P < 0.05), increased muscle mass by 25% (P < 0.02), and decreased liver mass by 7% (P < 0.02). Although the concentrations of most PCDD/F per gram of fat were slightly increased after clenbuterol treatment, the total amount of PCDD/F that remained in fat was reduced by approximately 30%. Muscle PCDD/F concentrations and total burden were decreased by clenbuterol. In contrast, clenbuterol tended to increase concentration, but not total burden of PCDD/F in livers. One congener known to be rapidly metabolized and excreted, 2,3,7,8-TCDF, was the exception to this increase, decreasing 40% with clenbuterol treatment. This was also the congener that showed the greatest reduction in both fat and muscle. Examination of the ratio of PCDD/F in liver and fat revealed that clenbuterol increased the liver's share of the body burden of PCDD/F, from 38 to 75%. In a remediation/disposal context, these findings would be beneficial if clenbuterol lowered the meat and carcass burden of PCDD/F to safe levels, requiring only livers to be disposed of as hazardous waste.

Technical note: epimerization of ergopeptine alkaloids in organic and aqueous solvents.

Purified ergopeptine alkaloids are often used in studies related to tall fescue toxicosis without regard to epimerization that occurs when ergopeptines are solvated. The objectives of this study were to measure the rates of alpha-ergocryptine epimerization to alpha-ergocryptinine at room temperature and at -40 degrees C, and to measure the rate of ergovaline epimerization to ergovalinine at 37 degrees C. Alpha-ergocryptine tartrate was stable (< 0.5% epimerization) in protic or aprotic solvents when stored at -40 degrees C for 20 to 52 d. At room temperature, alpha-ergocryptine epimerization in chloroform did not occur; epimerization was modest in acetone and acetonitrile (< 5%) but was substantial in methanol (78% by 38 d) and in a 70:30 water methanol mix (47% by 42 d). Ergovaline epimerization to ergovalinine occurred at 37 degrees C in 0.1 M phosphate buffers (pH 3, 7.5, and 9) in 9% aqueous solutions of fetal bovine serum (FBS), and in water, methanol, and acetonitrile. The degree of epimerization at 37 degrees C was solvent-dependent. Epimerization rates with respect to time were roughly linear in phosphate buffer (pH 3 only), water, methanol, and acetonitrile; epimerization rates resembled first-order kinetics in phosphate buffers (pH 7.5 and 9) and in the presence of FBS (pH 3, 7.5 and in Dulbecco's culture media). Epimerization equilibria (48 to 63% ergovaline) were reached within approximately 1 to 19 h. Results from this study indicate that researchers conducting studies with purified ergopeptines should carefully control the storage conditions of solvated ergopeptines and measure isomeric composition under the actual experimental conditions used in experiments.

Response of C2C12 mouse and turkey skeletal muscle cells to the beta-adrenergic agonist ractopamine.

The effects of ractopamine (RAC) and ractopamine stereoisomers (RR, RS, SR, and SS) on cyclic AMP (cAMP) production, total protein, and DNA concentrations in mouse skeletal muscle cells (C2C12) were evaluated. The RAC (10 microM) caused an approximately 30% increase in cell number, protein, and DNA concentrations in myoblasts after 48 h; no differences were found in myotubes. The RAC-stimulated increase of these variables in myoblasts was blocked by the presence of equimolar concentrations of propranolol. At a later passage, myoblasts failed to exhibit an increase in cell number, protein, or DNA upon exposure to RAC. Both myoblasts and myotubes increased cAMP production in response to 10 microM RAC. The RAC isomers ranked RR >> SR > RS approximately SS in ability to stimulate cAMP production, with essentially no response to SS. The SR produced about 50% of the RR response. Coincubation of propranolol (10 microM) and RAC (10 microM) prevented RAC-stimulated cAMP production in myotubes but not in myoblasts (approximately 35% of cAMP produced by RAC alone). Turkey satellite cells (derived from biceps femoris of 12-wk-old toms) produced essentially no increased cAMP when exposed to 10 microM RAC stereoisomers. Stability of RAC was evaluated under laboratory storage and culture conditions. The RAC was stable for more than 4 mo when stored in deuterated DMSO (>98% purity) at room temperature or in aqueous solutions at -80 degrees C, as determined from sequential nuclear magnetic resonance studies. Radiolabeled RAC was incubated for 72 h in the presence of serum-containing medium, with or without C2C12 cells. Ninety-eight percent of the parent compound found in the medium at time zero was present in the medium as parent at the end of 72 h. The cellular cAMP response to RAC through beta-adrenergic receptors seems to be stereospecific. If the state of myoblasts and myotubes in vitro reflects the in vivo state, then the ractopamine effect in vivo on cellular processes (including cell division and protein and DNA accumulation) may be independent of beta-adrenergic receptors in muscle.

Identification of the major urinary and fecal metabolites of 3,5-dinitrobenzamide in chickens and rats.

Colostomized chickens given oral doses of 3,5-dinitrobenzamide (nitromide) cleared nitromide predominantly through the urine (58% of dose) and feces (21% of dose). Rats cleared 52% of nitromide via urinary excretion and 44% via feces. Major urinary metabolites for both chickens and rats include: 3-amino-5-nitrobenzamide, 3-acetamido-5-nitrobenzamide, 3-acetamide-5-aminobenzamide, and 3,5-diacetamidobenzamide. The major fecal metabolite in chickens was 3-acetamido-5-nitrobenzamide (67% of fecal 14C) and 3-acetamido-5-aminobenzamide in rats (approximately 50%).

Endoplasmic reticulum lumenal proteins of rat mammary gland. Potential involvement in lipid droplet assembly during lactation.

Intracellular distribution of selected reticuloplasmins, soluble proteins of the endoplasmic reticulum lumen, in rat mammary gland was investigated during pregnancy, lactation, and involution. During lactation the levels of the calcium binding protein calreticulin, and of protein disulfide isomerase, were elevated. Endoplasmic reticulum was as efficient as Golgi apparatus in sequestration and accumulation of Ca2+ from surrounding medium, as suggested from in vitro experiments with isolated cell fractions. Both protein disulfide isomerase and calreticulin were present in cytosol from homogenates of mammary gland prepared under mild conditions. Protein disulfide isomerase was abundant in intracellular lipid droplet precursors of milk lipid globules. Calreticulin and immunoglobulin binding protein (BiP, GRP 78) were associated with lipid droplets. Glucose-regulated protein (GRP 94) was not detected in association with intracellular lipid droplets. Milk lipid globule membrane lacked more than barely detectable quantities of protein disulfide isomerase, calreticulin, and immunoglobulin binding protein, suggesting that these proteins are lost from intracellular lipid droplets before or during their secretion as milk lipid globules. Immunocytochemical localization confirmed the presence of protein disulfide isomerase or calreticulin on intracellular lipid droplets and in non-endoplasmic reticulum regions of cells.

Polyamine and polyamine analog regulation of spermidine/spermine N1-acetyltransferase in MALME-3M human melanoma cells.

In MALME-3M human melanoma cells the polyamine analog N1,N12-bis(ethyl)spermine (BESPM) suppresses the key polyamine biosynthetic enzymes, ornithine and S-adenosylmethionine decarboxylase, and increases the polyamine catabolizing enzyme, spermidine/spermine N1-acetyl-transferase (SSAT) by more than 200-fold. In the present study increases in SSAT activity in MALME-3M cells treated with 10 microM BESPM were found to be accompanied by a substantial (up to 45-fold) accumulation of SSAT mRNA. By Northern blot analysis three RNA transcripts were found to hybridize with the coding region of human SSAT cDNA: a minor high molecular weight (approximately 3.5 kilobases) species designated form A and two lower molecular weight species designated forms B and C (approximately 1.5 and approximately 1.3 kilobases, respectively). Form A increased uniformly during BESPM treatment and was most obvious in nuclear RNA preparations. On the basis of size similarity to the transcribing region of the gene and hybridization with the coding region of SSAT cDNA and its prevalence in nuclear mRNA preparations, form A is thought to represent precursor SSAT RNA. Form C is present in control cells and increases steadily during treatment, whereas form B increases transiently during early treatment (1-3 h). By RNase H digestion assay, form B was found to have a 200-base pair longer poly(A) tract and as such may represent a precursor to form C. Accumulation of SSAT mRNA was found to be a result of increased gene transcription and stabilization of SSAT mRNA. Nuclear run-on studies indicated a 2-4-fold increase in the transcription rate of the SSAT gene. As indicated by actinomycin D studies, the SSAT mRNA half-life increased with BESPM treatment from 17 to 64 h. The natural polyamine, spermine, also increased SSAT mRNA (5.5-fold at 24 h) and behaved similarly to BESPM in inducing the appearance of the same three transcript forms. The polyamine was much less effective than the analog at increasing enzyme activity. Lowering intracellular polyamine pools with inhibitors of biosynthesis decreased basal SSAT mRNA levels by at least 70% indicating, that the gene can be down-regulated as well as up-regulated by polyamines. These findings indicate that SSAT represents a unique example of gene expression being positively influenced at the RNA level by polyamines and their analogs.

Regulation of spermidine/spermine N1-acetyltransferase by intracellular polyamine pools. Evidence for a functional role in polyamine homeostasis.

Through its role in polyamine acetylation and the back-conversion pathway, spermidine/spermine N1-acetyltransferase (SSAT) has the potential to control intracellular polyamine pools by facilitating their catabolism and/or excretion. The possibility that the enzyme is subject to regulation by intracellular polyamine pools was investigated in MALME-3 human melanoma cells. Increases in intracellular polyamine pools by treatment with 3 microM exogenous spermidine or spermine for 48 h caused SSAT activity to increase 111% and 226%, respectively, and SSAT-specific mRNA to rise 19% and 66%, respectively. Decreases in polyamine pools by treatment with inhibitors of polyamine biosynthesis caused SSAT activity to decrease by 46% and mRNA to fall by 89%. Both SSAT activity and mRNA were more sensitive to changes in spermine than spermidine. The identification of a positive regulatory relationship between SSAT and intracellular polyamine pools further implicates this enzyme in a proposed model for polyamine pool homeostasis.

Differential effects of the spermine analog, N1, N12-bis(ethyl)-spermine, on polyamine metabolism and cell growth in human melanoma cell lines and melanocytes.

We have previously found that in one of two human melanoma cell lines, potent increase in the polyamine catabolizing enzyme, spermidine/spermine N1-acetyltransferase (SSAT), correlate with growth sensitivity to the spermine analog, N1, N12-bis(ethyl) spermine (BESPM). Herein, we examine the generality of this SSAT response among seven human melanoma cell lines (LOX, SH-1, STO-1, HO, PANUT-3, MALME-3 and Ebey) and normal melanocytes and further evaluate its possible correlation with growth sensitivity. Following treatment with 10 microM BESPM for 48 hr, SSAT activity among the various cell lines increased from basal levels of 20-90 pmol/min/mg to levels ranging from 170 to 30,470 pmol/min/mg. Five of the seven cell lines and melanocytes induced SSAT activity to levels to greater than 2,500 pmol/min/mg and three of these, to levels greater than 10,000 pmol/min/mg. When ranked according to SSAT responsiveness (LOX less than SH-1 less than STO-1 less than HO less than PANUT-3 less than MALME3 less than Ebey), there was a general correlation among the cell lines with growth sensitivity. Antiproliferative effects ranged from slowing of cell growth in the less SSAT responsive lines (LOX, SH-1) to total cessation of cell growth or overt cytotoxicity in the more potently SSAT responsive lines (MALME-3, Ebey). The polyamine biosynthetic enzyme activities, ornithine and S-adenosylmethionine decarboxylase, were similarly suppressed in all cell lines, presumably via analog activation of inherent regulatory mechanisms. Polyamine pool reduction occurred to a greater extent than predicted in cell lines where SSAT was increased to greater than 2500 pmol/min/mg suggesting a possible role for the enzyme in enhancing polyamine excretion and/or catabolism. The occurrence of potent SSAT induction among several human melanoma cell lines and the growth sensitivity of these same lines to BESPM suggests that the enzyme response may represent a determinant of drug action in this particular malignancy.

Comparative growth of normal and malignant mouse mammary epithelium cultured serum-free on a biomatrix from preadipocytes.

Growth of normal and malignant mouse mammary epithelial cells (MMEC) on a biomatrix of substrate-attached material from 3T3-L1 preadipocytes was evaluated to devise culture conditions that are suitable for transformation studies but do not involve embedding cells in a gel. The biomatrix was prepared as described by Levine and Stockdale, and serum-free medium contained bovine serum albumin, insulin, progesterone, prolactin, and linoleic acid. Each cell type produced a distinctive pattern of colony architecture in this culture system. Cells from virgin mice (vMMEC) usually formed elaborate, three-dimensional structures resembling ducts and alveoli; cells from pregnant mice (pMMEC) grew as flat monolayers; and tumor cells grew in multilayered clusters. Cell growth was monitored by an assay for succinate dehydrogenase. Similar growth rates were found through Day 8 in cultures of vMMEC and D2 carcinoma cells. Growth of vMMEC slowed thereafter, whereas tumor cells typically continued growing through Day 14 to 18. Increase in cell number during 18 days in culture was 3-, 7-, 9-, and 11-fold for cells from pregnant and virgin mice, BALB/cfC3H and D2 carcinomas, respectively. The percent cells in S phase on Day 2 of culture was 9% for pMMEC, 4 to 11% for BALB/cfC3H tumor cells, 20% for vMMEC, and 24% for D2 tumor cells. Thus, this culture system promotes extended growth of MMEC and offers several advantages over embedding cells in a collagen gel. It may therefore be applicable to in vitro transformation studies with MMEC.

Effects of dietary calcium and age on parathyroid hormone, calcitonin and serum and milk minerals in the periparturient dairy cow.

Ten pregnant heifers and 10 pregnant cows (three or more pregnancies) were assigned to groups of five and fed either the recommended (low) amount of calcium or excess (high) calcium in their diet for 4 wk before parturition to determine the influence of prepartum calcium intake and age on hormonal control of peripartum calcium homeostasis. After parturition all groups received a diet with calcium adequate for lactation. Jugular blood samples from 21, 14, 7, 6, 5, 4, 3, 2, and 1 d prepartum through 0, 1, 2, 3, 5, 7, 14 and 21 d postpartum were assayed for concentration of parathyroid hormone, calcitonin, calcium, magnesium and phosphorus. Heifers and cows receiving high calcium diets had higher calcium and lower parathyroid hormone in blood serum before parturition than animals receiving the low calcium diets. Cows, but not heifers, fed high calcium diets exhibited severe hypocalcemia at parturition, remained hypocalcemic for 3 d and had low serum calcitonin. Regardless of dietary group, concentrations of parathyroid hormone and magnesium in serum increased after the first week of lactation. Feed intake during lactation, corrected for metabolic body weight, was similar for both dietary treatments and ages. Milk production per kilogram metabolic body weight was highest during the first week of lactation for cows fed low calcium diets before parturition. There was no correlation between hypocalcemia and loss of calcium in colostrum or milk. Feeding low dietary calcium to cows in the prepartum period was effective in the prevention of severe hypocalcemia at parturition. In contrast, dietary treatment of heifers had no effect on serum calcium concentration at parturition.(ABSTRACT TRUNCATED AT 250 WORDS)